FTIR Spectroscopy of a Viral Rhodopsin, OLPVR2

Microbial rhodopsins in giant viruses are classified into two major classes: VR1 and VR2. Organic Lake Phycodnavirus Rhodopsin 2 (OLPVR2) belongs to the VR2 family, while OLPVR1 is a viral channelrhodopsin. OLPVR2 is known to form a pentamer, and it was proposed that OLPVR2 is a light-gated ion channel, whose ion pathway is not inside the monomer but in the pentameric center. OLPVR2 possesses a water-containing pentagonal cluster structure in the retinal Schiff base region, which is very similar to that of the OLPVR1 and bacteriorhodopsin (BR), a light-driven proton pump. In this study, we applied low-temperature FTIR spectroscopy to OLPVR2 at 77 and 170 K and compared the results with those of OLPVR1 and BR. Photoexcitation of OLPVR2 leads to isomerization from the all-<i>trans</i> to 13-<i>cis</i> retinal, and a red-shifted K intermediate is formed with the distorted chromophore, as well as in OLPVR1 and BR. On the other hand, OLPVR2 does not show any spectral changes for protonated carboxylic acids at 77 K, but a new band appeared at 170 K. We observed stronger hydrogen out-of-plane (HOOP) vibrations at 170 K than at 77 K, suggesting that chromophore distortion is more enhanced at 170 K. FTIR analysis of protein-bound waters revealed that the strength of the water hydrogen bond is much weaker in OLPVR2 and OLPVR1 than in BR, and lack of strongly hydrogen-bonded water molecule is characteristic of viral rhodopsins. In viral rhodopsins, the water molecule bridging the Schiff base and counterions forms hydrogen bonds to two counterions equally, unlike the case in BR. The mutation study further implied that the counterion effect is larger for the aspartate in the C helix in OLPVR2 and for the aspartate in the G helix in OLPVR1.